The recent identification of the cystic fibrosis (CF) gene, the cystic fibrosis transmembrane conductance regulator (CFTR) gene (Riordan JR et al., Science 1989;245:1066-73), revealed that the gene belongs to a family of membrane transport molecules that includes the multi-drug resistance genes. Approximately 70% of CF chromosomes contain a deletion of 3-base pairs resulting in the loss of a phenylalanine codon at amino acid position 508 (delta F508). The focus of this project is to identify new mutations in this gene that comprise the remaining 30% of CFTR gene mutations. Using oligonucleotide primers based on the sequence of the CFTR gene, we have used the polymerase chain reaction to amplify several coding exons. These regions have been examined from 110 patients that contain 127 chromosomes without the common CF mutation (delta F508). Eight additional mutations have been identified in this group, in a total of four different exons. Most of the mutations were initially identified using an assay for single-- stranded conformation polymorphisms. All mutations were subsequently characterized by direct sequencing of the amplified DNA and can be assayed by restriction enzyme digestion or allele-specific oligonucleotide hybridization. The mutations fall into two classes: (1) one or two nucleotide insertions and deletions that introduce termination codons into the gene and are predicted to result in severely truncated protein products, and (2) point mutations in the putative membrane-spanning domains that replace charged amino acids with nonpolar residues. Sequencing of the membrane-spanning region from several species demonstrates that this region is highly conserved across species.